This application claims the priority of Korean Patent Application No. 2002-70141, filed on Nov. 12, 2002, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a method for detecting a polymerase chain reaction (PCR) product using an electrical signal.
2. Description of the Related Art
A representative biosensor includes an enzyme sensor and an immune sensor using an immune response of the immune system. However, as a considerable amount of DNA information is obtained by the completion of the human genome project, and thus, potential and expectation of an early discovery and treatment of human genetic diseases increase, a DNA chip is rapidly emerging as another type of biosensor. Presently, active research areas in the development of biosensors are the possibilities of obtaining a low manufacturing cost, high speed, accuracy, easy handling property, and miniaturization (portability). In this regard, a DNA sensor for detecting a human disease at an early stage by using DNA information must be also developed toward satisfaction of the above requirements to strengthen an international market competitiveness.
One of essential genetic assays is a PCR DNA assay, which has been widely used in clinical, biological, and genetic laboratories since it was invented at the end of the 1980s.
A traditional PCR shows the qualitative results of amplified DNA by a gel electrophoresis at the end-point of the PCR reaction, but has many problems such as inaccuracy of the quantitative detection of DNA. Therefore, a Real-Time PCR was developed to allow for the quantitative detection of amplified DNA by detecting the intensity of fluorescent light, which is in proportional to the concentration of the amplified DNA, using an optical detection system.
A quantitative assay of DNA is essential for studies of disease treatments and DNA expression. For example, in order to ensure a successful medicinal therapy for patients infected with hepatitis B-type virus (HBV), the drug resistance of HBV must be tested by periodically detecting the concentration of HBV in the blood plasma using a Real-Time PCR.
A conventional Real-Time PCR requires many optical devices such as a laser source, a micromirror, a microscope, and a filter, and an expensive fluorescent dye. In addition, because a conventional Real-Time PCR chip is based on the principle of detecting fluorescent light, there are many disadvantages in terms of miniaturization (on a chip) and economical efficiency.
In order to solve this problem, an effort was made to electrically detect DNA using capillary electrophoresis (CE) [Christa L. Colyer et al, Journal of Chromatography A, Volume 781, Issues 1–2, 26 Sep. 1997, pp. 271–276; F. Laugere et al, Sensors and Actuators B: Chemical, Volume 83, Issues 1–3, 15 Mar. 2002, pp. 104–108; Pumera et al., Anal. Chem., 2002, 74(9), pp. 1968–1971). This method allows for a qualitative assay, but has many problems for a quantitative assay. In addition, transfer of PCR products to a CE detection system using a micro-channel after the completion of PCR is a laborious process and a high voltage is required. Therefore, requirements of economical efficiency and miniaturization are not satisfied.
Miles et al. filed a U.S. patent application, which has been published under U.S. Patent Application Publication No. 2002/007254A1, based on the concept that as the concentration of DNA increases during PCR, impedance decreases and conductivity increases. However, the Miles' concept that impedance decreases as the concentration of DNA increases during PCR is contrary to the fact demonstrated by the present inventors. Therefore, this patent application comes from a misunderstanding of the mechanism of PCR reaction.
During PCR, dNTPs are dissociated into dNMPs and many diphosphates. At the same time, dNMPs are polymerized into DNA using a primer supplementary to a template DNA. In this case, the disclosure of Miles et al. considers only the electrical mobility of diphosphates as a byproduct. However, considering an entire electrical mobility which decreases by dNMPs, a primer, and a template, all of which have electric charges during DNA synthesis, as the concentration of DNA increases, impedance of PCR increases due to the decrease of the electrical mobility. Furthermore, the Miles' invention is concerned with a PCR chip for end-point detection not a Real-Time PCR chip. In addition, there is a problem in that an ionically-labelled probe must be used to detect an amplified PCR product.